Insects of forensic importance: habitat, life cycle and succession; Thanatological relationship to insects; Post-mortem interval and entomological relevance; Factors that influence succession; Legal admissibility of forensic entomology; Crime scene documentation; Collection of insects from the body; Analysis of entomological evidence; Report writing; Forensic entomotoxicology
2. Forensic entomology is the application of the study of arthropods
(order Arthropoda), including insects, arachnids (spiders and their
kin), centipedes, millipedes, and crustaceans, to criminal or legal
cases.
Typically, the use of insects and their life cycles helps to establish a
PMI, which is an estimate of how much time has passed since a
person died.
Often, insects are the first to find a corpse, and they colonize it in
a predictable pattern. Forensically important conclusions may be
drawn by analyzing the phase of insect invasion of a corpse or by
identifying the life stage of necrophagous (dead-flesh eating)
insects found in, on, or around the body.
FORENSIC ENTOMOLOGY
3.
4. Necrophagous insects (insectsfeedingonthecorpse)
• Blow flies (Calliphoridae) andFlesh Flies(Sarcophagidae): importantinsectsfordetermination of PMI(usethecorpseforreproduction)
• Scuttleflies (Family Phoridae) :Discover wellhidden,covered, wrapped, orburied bodies
• Skipper flies (Family Piophilidae): associated withbonesofcarcass orcorpse
• Carpet or hide beetles (Family Dermestidae): Necrophagous beetles feed on skin and hair of corpse in the late stages of
decomposition.
Parasitoidsandpredators
• Rove beetles(Family Staphylinidae): preyon blowflyand flesh flylarvae during early stagesof decay.
• Wasp( Nasoniavitripennis):parasitoids ofnecrophagous dipterapuparia
Omnivorousspecies (Insectsthatfeedonboththecorpseandassociatedarthropods)
• carrion beetles,ants, andyellowjacket wasps (retard therate ofcarcass removal bydepleting populations of necrophagousspecies)
Adventitiousspecies (Insects thatusethecorpseasanextension oftheirownnaturalhabitat)
• springtails, spiders,centipedes:use thecorpseas ashelter during veryearly (orlate) stagesofdecomposition.
• mayprovide information regarding themovementof abodyfromone environment toanother
7. Flesh Flies
(Family Sarcophagidae)
• mediumsized(2to14mm)andareover2,000species
• mostspeciesoccurringeitherintropicalorwarmtemperateregions
• Theyfeedondecomposinghumanandanimaltissues,aswellasondecomposingvegetation.
• Theadultsareoftenfound onflowers,wheretheyareattractedtonectar,sapandhoneydew.
• However, the family’s Latin name means “flesh eating” and apparently refers to the larvae or
maggotsthattypicallyfeedoncarrion,excrementorexposedmeats.
• causemyiasis
• Appearance: bodies are bristly, the eyes (bright red in color in some sp. ),gray and black
longitudinal stripes on the thorax and checkerboardpattern on the abdomen (devoid of metallic
coloration)andprominentgenitalia(somespecies)
• Flesh flies are attracted to carrion under most conditions, including sun, shade, dry, wet,
indoors, and outdoors. They are frequently found on any decomposing tissues located within an
indoorenvironment.
• associatedwithcarcassesthroughoutboththeearlyandlatestages ofdecomposition.
• Flies of the genus Sarcophaga arrive concurrently with, or slightly after, the blow flies on
humanremains.
Source: Byrd & Tomberlin, 2019
9. SkipperFlies(FamilyPiophilidae)
• 2.5to4.5mmsizedflywithonly69species
• foundintemperateregions
• Appearance:Metallicblueorblackincolor
• habitats include carrion, human waste, bones, skin, and fur (dry in nature protein-rich food
sources).
• Themaggotsconsiderablylargerthantheadultsandusuallyrangefrom5to10mm.
• In cheese skipper (Piophila casei) “jumping” behavior as an effective “escape” mechanism,
and it is also utilized extensively during larval migration. However, they also move in the
moretraditionalcreepingmannerexhibitedbymostflylarvae.
Source: Byrd & Tomberlin, 2019
10. HumpbackedFliesorScuttleFlies
(FamilyPhoridae)
• Smallsize(1.5to6mm)andmorethan2,500speciesworldwide
• Appearance:humpbacked,maybeblack,brown,oryellowincolor
• Commonly associated with decaying plant matter and ubiquitous pests where live insect
coloniesaremaintained.
• The adult insect will run in a very characteristic swift and erratic manner, which has
earnedthemthecommonnameofscuttleflies.
• The larvae typically develop in any decomposing organic matter of human, animal, or
vegetativeinorigin.
• The puparia is dorsoventrally flattened, with a pair of horns or “breathing trumpets” on
anteriorend.
Source: Byrd & Tomberlin, 2019
11. SkinBeetles,Leather Beetles,HideBeetles,Carpet
Beetles,LarderBeetles(Family Dermestidae)
• smallbeetles(2to12mm)andover500species
• Appearance: rounded to oval in shape and covered with scales that may form
distinctiveandcolorfulpatterns.
• The carpet beetle species damage rugs, clothing, and furniture. Others, like the khapra
beetle, infest grains and inflict serious losses on stored products. Still others, like the
hide beetles, may ruin leather goods or destroy irreplaceable museum specimens,
especiallymounted insects.
• Skin beetles are of forensic importance reducing a human body to a skeleton in only
24days.
• The larvae range from 5 to 15 mm and covered with tufts of long, dense hair found on
humancorpsesduringthedryandskeletalstages ofdecomposition
• Theadultsarecannibalisticandwilleatyounglarvaeandpuparia.
• These beetles can be found in indoor situations on bodies throughout the year, but are
mostactiveduringthewarmermonths.
• The presence of dermestid beetles or their sawdust-like frass (fecal material) is often
anindicationthatconsiderable timehaselapsedsincedeath.
Dermestesmaculatus (Hideorleatherbeetle)
12. RoveBeetles(Family
Staphylinidae)
• Size(1to25mm)andover47,700species.
• Feedondecomposing animaltissue,plantdebris,andfungi.
• Appearance : slender, elongate, and have very short wing covers or
elytra. The head, thorax, and wing covers make up the first three
sections and are approximately equal to each other in size. The fourth
sectionistheexposedabdomen, which isroughly equal toall ofthefirst
threetogether.
• larvae are typically long, slender, pale in color, and may have a darker
head.
• Larvae and adults are typically quick moving and predacious on
smallerinsects.
• The adults are strong flyers and often run about with the abdomen
raisedintheair(faking stingingaction)
Creophiliusmaxillosus
(Hairy rovebeetle)
13. ClownBeetles(Family
Histeridae)
• Smallsize(upto10mm)andlargefamilyofover3,000species
• Appearance: small , rounded, shiny beetles that are black or sometimes
metallicgreen.
• Common on carrion and excrement, as well as on fungi and decaying plant
material.
• On carcasses, they tend to stay concealed in the soil underneath during the
daylighthours,becomingactiveatnight.
• Both the larvae and adults are predacious and feed readily on maggots and
flypuparia(larvaeofdermestidbeetles).
Saprinus pennsylvanicus
(Clownbeetle)
14. Checkered Beetles(Family
Cleridae)
• Sizes(3to12mm)and 3,500species
• Appearance: The bodies covered with bristly hairs and are often
brightlycolored.
• Both the larval and adult are predacious (immature stages of various
wood-boringbeetles)
• frequently found on flowers and common visitors to decomposing
animalmatterinthelater,drierstagesofdecomposition.
Necrobiarufipes
(Red-leggedhambeetle,coprabeetle)
15. References/Suggested Readings:
Byrd, J. H., & Tomberlin, J. K. (Eds.). (2019). Forensic entomology: the utility of arthropods in legal investigations.
CRCpress.
Rivers,D. B., &Dahlem, G. A. (2014). Thescienceofforensicentomology.JohnWiley&Sons.
19. Arthropod evidence can be used to link a suspect to a victim
• Finding the same kind of arthropod at the site of a victim body as one associated with the clothing or vehicle
of a potential suspect has helped to incriminate individuals in several murder cases.
• Case : Investigators at the scene where the victim was found developed very itchy skin rashes, with spots on
the ankles, waist, and buttocks. Similar lesions were seen on a suspect in the case. Further analysis and field
study indicated that these lesions were caused by the bites of the chigger Eutrombicula belkini (rare mites),
a species found in very limited geographic areas, which included where the murder victim was discovered.
Arthropod evidence can be used to determine body disturbance
• There are reported differences in the species of insects involved with the decomposing corpse in different
habitats and environments.A careful examination can reveal species variation, as species associated with one
type of habitat present on a corpse is found to be different from those when the corpse is transported after
death.
Arthropod evidence can be used to determine presence and position of wounds
• accumulations of maggots or other insects feeding on anatomical bodyparts other than at the common
oviposition sites at body orifices may suggest the presence of wounds. The insect distribution may be used
only to corroborate other evidence, such as pathological or physical evidence of stab wounds, cuts, gunshot
wounds, or abrasions.
20. References/Suggested Readings:
• Krinsky, W. L. (2019). Forensic entomology. In Medical and Veterinary Entomology (pp. 51-60). Academic
Press.
• Joseph, I., Mathew, D. G., Sathyan, P., & Vargheese, G. (2011). The use of insects in forensic investigations:
An overview on the scope of forensic entomology. Journal of forensic dental sciences, 3(2), 89.
21. Neglect of Elderly and Children
• The use of insects as indicators of abuse, or alleged abuse, has been long known.
• Due to the fact that wounds of living persons (Myiasis) are a potential target for the
same flies that colonize corpses and decomposing tissues.
• The misconduct of elderly people and children currently becomes a severe problem.
• Forensic entomology can give important insights into the dynamics, the amount, and
the final state of bodily care that was given to the neglected person.
• At the same time, forensic entomology helps to excuse care givers who did actually do
their duty whilst maggot infestation of a person’s wounds occurred during a normal
interval of non-visits.
22. Case 1: Clean apartment with dead Muscina stabulans
• An elderly woman was found dead in October 2002 in her
third floor apartment in urban, Germany (Fig. 1). The
apartment was very clean except of the bath room in which a
bath tub had been filled with water and clothing.
• Entomological evidence : larvae & dead adult flies
(Muscina stabulans) on a window sill pointing. Blowflies
absent in any developmental stage.
• Interpretation: PMImin was estimated around 3 weeks
indicating towards misconduct of the paid professional care
giver who was supposed to check for the women every week.
• The care giver, however, claimed that she had called the
women 2 weeks ago to check on her; the dead women
allegedly rejected any visits (old woman mentally unstable and
behaving ‘‘difficult’’ against everybody).
• In clear contrast to the entomological findings, the prosecution
assumed that the care giver tried her best, and therefore, the
D.A.’s (District attorney) office closed the case.
Fig. 1. Note that the eyes are intact whilst the lower part of
the body is partially decayed due to influence of feces and
urine/bacteria. Upper part of body mummified (dried out).
Source: Benecke et al. 2004
23. • In September 2002, an old woman was found dead in her
apartment in an urbanized town in Western Germany.
The apartment was in a bad condition, and even the
landlord had noted in January 2002 that renovations were
urgently necessary due to wet spots in the walls. He also
noted the presence of ‘‘small flies’’. The women did not
clean her toilet appropriately, and in the washbowl, wet
clothing was found
• Entomological evidence: Her foot was wrapped in a
plastic inside, numerous larvae of Lucilia sericata
(Blowflies).
• Interpretation: PMImin interval as 2 days. The age of
the maggots was estimated from their size (11 mm) as
approximately 4 days at recorded outside temperatures
of 20 degree Celsius. However, judging from the deep
tissue loss at the foot, it was discussed that most likely,
the maggots had been feeding on the living women for
at least a week whilst she was still alive but then left the
bag to pupate elsewhere.
Fig. 3. (a) Regular post mortem state of corpse, no external signs of
violence; (b) Influence of maggots on live tissue.; (c) non-functional
toilet aided in establishing fly population before wound was present.
Case 2: Deep tissue loss at foot
Source: Benecke et al. 2004
24. • In the following case the mother had left the apartment at an unknown
time. Due to very severe drug use and an unstable lifestyle (reported street
prostitute), she had no recollection of when she was in the apartment for
the last time. During the investigation, it also became important to
determine the total time the child may have been left alone, and thus
neglected, because social workers were accused of neglect by the
prosecution.
• Entomological evidence: Under the diaper and on the surface of the skin
of the deceased child (anal-genital area) third-instar larvae of the
Muscina stabulans (false stable fly) and Fannia canicularis (the lesser
house fly ) . From the face, larvae of the Calliphora vomitoria (bluebottle
fly).
• Interpretation: F. canicularis adults are attracted to both feces and
urine. . It is commonly reported that C. vomitoria maggots are typical
early inhabitants of corpses.
• From the developmental times of the flies, it was estimated that the anal-
genital area of the child had not been cleaned for about 14 days, with a
total estimated range of time from 7 to 21 days, and that death occurred
only 6 to 8 days prior to discovery of the body.
• In the first trial, this led to a conviction not only of the mother but also of
one of the social workers.
CASE 4: CHILD NEGLECT
Fig 5 On the skin surface under
the diaper (anal-genital area),
25. References/Suggested Readings:
Byrd, J. H., & Tomberlin, J. K. (Eds.). (2019). Forensic entomology: the utility of arthropods in legal investigations. CRC
press.(Chapter 20)
Benecke, M., Josephi, E., & Zweihoff, R. (2004). Neglect of the elderly: forensic entomology cases and
considerations. Forensic Science International, 146, S195-S199.
27. Introduction
• One of the most important factors in crime investigation of fatalities is to establish the cause of
death.
• This particular aspect may be difficult to ascertain when the body is recovered after it had been
badly decomposed or cases of delayed recovery (death due to drug abuse in isolated places or
suicide or body deliberately concealed).
• In such a scenario, where conventional toxicological samples such as tissue, body fluids, and
internal organs have degraded or are no longer available, the only alternate reliable specimen
for forensic purposes are insects.
• Rationale: Insects are of forensic significance as they utilize the dead body as food source for
their larvae. While actively feeding on cadaveric tissue, xenobiotics such as drugs and other
toxic substances that are present in the tissue gets transferred to the metabolic system of
larvae.
• In such cases, entomotoxicology would be a useful tool in solving poisoning cases when dead
bodies are recovered after prolonged period of time
28. Source: Chophi et al. 2019
Various drugs and toxic substances reported in forensic entomotoxicology.
29. Insects as toxicological sample
• The organic materials of entomotoxicological interest which can be analysed are:
• larvae
Pupae
adult insects
puparial cases
exuviae (cast beetle skins)
beetle faecal material (frass)
fly predators and scavengers
• The insects most frequently involved in toxicological analyses are true flies (Diptera) and beetles
(Coleoptera).
30. Sample collection and preservation
• Sample collection : entomological material from entire body of the corpse (and up to 10m
surrounding area)
• Preservation: eggs in 70–95% ethanol, killing larvae at 80 °C hot water for 30s and storing in 70–
95% ethanol, storing pupae in container at 2°C–6°C temperature with punched holes in lid, killing
of adult flies in vial freezed at −20 °C and storing the dead specimen in 70–95% ethanol.
• Preservation for toxicological analysis: specimens are preserved at −4°C and the analysis are
carried out in the same manner as human tissues or fluids of toxicological interest.
• Storage of specimen: done under dry conditions at −20 °C in order to ensure drug stability.
31. Toxicological analysis
• Different extraction techniques
such as liquid-liquid extraction
and solid phase extraction are
employed for extracting various
poisons and drugs according to the
chemical features of the
substances to be detected.
• Solid phase extraction is reported
to give the best organic toxicant
purification from aqueous extracts
of entomological specimen.
• Entomotoxicology study samples
R&D: different animal models
(rabbits, rats), and meat substrate
spiked with drugs
Source: Chophi et al. 2019
Layout of the analysis of insect specimen for toxicological analysis.
32. Trends in the use of analytical techniques for entomotoxicological studies.
Source: Chophi et al. 2019
33. Limitations
• At the moment, the absorption, metabolism, elimination, and accumulation of drug and other toxic
substances in insects is not fully understood yet.
• The pharmacokinetics of drugs in insects depends on the species, the developmental stage, and on their
feeding activity as well.
• Analysis could also be influenced by factors such as drug stability, temperature and humidity.
• The major drawback of entomotoxicology field is the lack of interpretation of detected drug
concentration.
• PMI and Drug: drug may cause significant bias in estimation of post mortem interval when large amounts
of the drug are ingested prior to death. Thus wrong estimation of PMI can result if the presences of toxic
substances in tissues are not considered (eg. Methamphetamine was found to increase the growth rate of
larvae, Malathion decreased the growth rate of fly larvae)
34. References/Suggested Readings:
Chophi, R., Sharma, S., Sharma, S., & Singh, R. (2019). Forensic entomotoxicology: Current concepts, trends and
challenges. Journal of forensic and legal medicine.
35. Gut content: victim and suspect identifications
• Procedure used for victim and suspect identification by insect gut contents.
1. Evidence collection at the crime scene follows standard protocols, with special care taken to preserve
molecular and DNA evidence. Larvae collected at the crime scene should be preserved in ethanol, and
a subset should be reared to adults if possible.
2. The crop can be dissected and removed. The DNA is extracted from both the crop and its contents
using established DNA extraction methods (e.g., Qiagen DNeasy Blood and Tissue Kit).
3. Once the DNA extraction is complete, PCR is conducted to generate sufficient DNA for identification
of the insect and its food source.
36.
37. CASE DESCRIPTION:
In March 2008, a male headless corpse (Body I)
dressed only in underwear was discovered in a garden
in the city of Changsha (Fig 1A).
Soon after a separated skull (Body II), which was
wrapped in clothes, was found 500m away from the
former scene (Fig 1B).
Whether these two bodies were from the same
victim was difficult to judge because of their state
of decay.
Entomological evidence: Third instar maggots
Analysis of Entomological Evidence:
Collection of samples (maggots killed by boiling at
70 degree Celsius/Human tissue from coastal
cartilage)
Wash method and maggot dissection (20% bleach
sol. To clean environmental residues / 2 times rinse
water)
DNA extraction and amplification ( 16sRNA for
species identification rest of body part excluding crop,
STR analysis from Insect crop and human tissue)
38.
39.
40. Case Report
A badly burned body (pugilistic posture, with the feet and hands
missing) was discovered in a wooded area adjacent to a remote
country road.
Autopsy revealed an open head injury.
Because of the state of the corpse, soft tissues were not available
for further analysis, with the exception of a small burned
fragment of liver.
A woman was reported missing by her father, who claimed that his
daughter was abducted by several subjects 10 days earlier than the
remains were found. This man recognized the school graduation ring
recovered at the scene as belonging to his daughter. However, the
body was so badly burned that he was unable to identify it by facial
or other physical features.
Several attempts to obtain a genetic profile from the fragment of
liver recovered at autopsy were unsuccessful.
Entomological evidence:
The face and neck were extensively colonized by blowflies
(Diptera: Calliphoridae) and flesh flies (Diptera: Sarcophagidae)
fly larvae.
Analysis:
DNA extraction from the crops was performed using phenol–chloroform
extraction and for the alleged father DNA was extracted from a buccal
sample using a Chelex protocol for by STR analysis.
42. The role of insects in estimating the PMI
• several species of insect are attracted to carrion within a few minutes of death
• faunal succession is relatively predictable for specific stages of physical decomposition
• some species produce larvae whose development is tied to feeding on the corpse
• PMI could be estimated be estimated based on insect, specifically necrophagous fly, larval
development. The age of the oldest larva found on the body can be used for making A time
estimate of the association between the fly and body. This approach relies on working backward
from the developmental stage discovered to oviposition/larviposition. A time can then be
assigned (estimated) for how long development would have taken under the environmental
conditions associated with the crime scene.
43. Assumptions for using insects in calculating the PMI
1. Insects used for PMI estimations actually feed on the body to meet growth and developmental needs.
2. Adult females did not oviposit/larviposit on a live host (Myasis).
3. The insects are poikilothermic.
4. A linear relationship exists between temperature and insect growth in terms of immature stages, at
least for temperatures lying within the zone of tolerance for a given species and developmental stage.
5. The stage of insect development can be accurately determined ( also genus and species level
identification)
44. What is needed to calculate the PMI?
identification of the fly genus and species and age of development stage
experimental development data at relevant temperatures for insect of interest
base temperature or developmental threshold for each species of interest
temperature data from the crime scene
temperature data from a nearby weather station
calculation of accumulated degree days representing relevant stages of insect
development
calculation of accumulated degree days for the crime scene
45. Accumulated degree days for insect development
• Degree days are thermal units that reflect energy currency in the environment which an insect can
use for its own growth and development
ADD= time (days)×(temperature– base temperature)
ADH=time (hours) × (temperature–base temperature)
• The resulting units of measure are °D or °H, but are reported with regard to temperature scale. For
example, accumulated degrees days calculated based on a Celsius scale would have the units
ADD°C (as opposed to ADD°F).
• Time in this case is the length of time needed to complete a given stage of development at
temperatures comparable to the environmental temperature.
46. Calculate the accumulated degree days in °C (ADD°C) required for P. regina to complete the following
stages of development when this fly species is reared at 21.1 °C, with the lower threshold temperature for
development to take place being 11.4 °C:
(a) Egg stage
(b) First instar larva
(c) Second instar larva
(d) Third instar larva
(e) Pupa.
47. Corrected crime scene temperatures
What if the crime scene temperatures are not known?
The answer is to visit a meteorological weather station that is located close to
where the body was discovered.
Minimum/maximum temperatures can be averaged to provide an approximate daily
average temperature for a day of interest.
The relationship between C.S versus Met. Dept tempt. determined by measuring
temperatures at the crime scene from the time of discovery for 4–5 days (generally
such temperatures are measured at approximately 1.2 m or 4 feet from the ground
surface )
Simple linear regression is performed, and based on the resulting r-value
(Correlation coefficient) the strength of the relationship between the two sites can
be made.
High r-values are indicative of the weather station temperatures being reflective of
the crime scene temperatures, while low values suggest that the meteorological
data do not approximate the crime scene temperatures well.
In the latter scenario, use of such data will lead to over- or under-estimations of the
PMI.
If there is a strong relationship between the two sites, then the regression equation
can be used as a correction factor
Tc = (5 / 9) × (Tf –32) (COVERSION FROM DEGREE CELCIUS TO DEGREE FAHRENHEIT)
Figure: Determination of relationship between
crime scene temperatures and those from a
meteorological weather station. The regression
equation is used to calculate corrected crime
scene temperatures.
48. Use the following information and Table 2 to calculate
the environmental energy available for larval
development of Calliphora vicina from June 28 to July
11. Assume that developmental data are available or
26.1 °C, the calculated base temperature is 3 °C, and
use the corrected temperature regression of y = 0.93x +
2.0. Construct a table to organize the information.
49. References/Suggested Readings:
Rivers, D. B., & Dahlem, G. A. (2014). The science of forensic entomology. John Wiley & Sons. (Chapter
12)
Research Articles for additional readings:
Bala, M., & Sharma, A. (2016). Postmortem interval estimation of mummified body using accumulated degree hours (ADH) method:
a case study from Punjab (India). J. Forensic. Sci. & Criminal Inves, 1, 1-5.
Sharma, A., & Bala, M. (2016). Case Study and PMI Estimation of Male Corpse from Ludhiana, Punjab, India: An Implication of
ADH Method. Indian Journal of Forensic Medicine & Toxicology, 10(2), 28-33.
Hu, G., Wang, M., Wang, Y., Liao, M., Hu, J., Zhang, Y., ... & Wang, J. (2020). Estimation of post-mortem interval based on insect
species present on a corpse found in a suitcase. Forensic science international, 306, 110046.
Editor's Notes
For the poikilothermic ectotherms the internal body temperature cannot be maintained by metabolic heat and thus reflects ambient environmental conditions
zone of tolerance or thermal tolerance range: The range of temperatures over which insects can maintain metabolic processes and survive
Critical thermal maximum: temperatures above the upper thermal limit
Critical thermal minimum: temperatures the lower thermal limit
**Both Critical thermal max. or min. conditions initially evoke inhibition of cellular reactions, thereby retarding most aspects of growth and development. If necrophagous species are exposed for a sufficiently long period injury or death may be the result.
The base temperature or developmental threshold is the lowest temperature at which insect development can occur.
Concerns:
1. Development rate is dependent on available nutrition.
Developmental rate is also influenced by size,density and/or volume of a maggot mass (small maggot mass; slow larval development whilst too large maggot mass; overcrowding competition and heat stress slowers larval development.)
Determination of adult eclosion can also be problematic Usually there is a 3–5 day window of adult emergence for most species of calliphorids and sarcophagids.